Genome Scale Reagents and Services for Functional Studies January 21st - 22nd, 2014 Biomedicum...

Genome Scale Reagents and Services for Functional StudiesJanuary 21st - 22nd, 2014Biomedicum Functional Genomics Unit (FuGU)

RECOMBINANT VIRUS PRODUCTS AND qRT-PCR SERVICES AVAILABLE AT FuGU

• Established in 2006, operates under the national Biocenter Finland infrastructure network

• Co-directed by Outi Monni (genome profiling) and Juha Klefström (recombinant virus technologies)

• Located at Meilahti campus of the University of Helsinki

– Biomedicum Helsinki 1 building

• Services include e.g. next-generation sequencing, microarrays by four commercial platforms, recombinant virus services and genome-scale reagents for gene knockdown

January 21st-22nd, 2014 FuGU 2

Biomedicum Functional Genomics Unit

Recombinant Virus Products and Services Available at FuGU

• Genome scale shRNA library for RNA interference -mediated gene silencing

• Both human and mouse TRC1 shRNA libraries available– developed by the RNAi Consortium at the Broad Institute of MIT and Harvard

– ~159,000 pre-cloned shRNA constructs targeting ~16,000 annotated human and ~15,950 mouse genes - in pLKO.1 lentiviral vector backbone

• on average, 3-5 constructs per gene• Two in five clones will typically provide at least 70 % knockdown

• TRC1 shRNA library controls:– Empty pLKO.1 vector– Non-target (scramble) shRNA in pLKO.1 backbone– TurboGFP in pLKO.1 backbone– eGFP shRNA in pLKO.1 backbone

• same controls for human and mouse libraries


The RNAi Consortium (TRC) shRNA library

TRC Library Database:http://www.broadinstitute.org/rnai/public/

*shRNA (short hairpin RNA): Mimics miRNA by forming a hairpin structure, utilizes the endogenous RNAi pathway both in the nucleus and in the cytosol. Can be integrated into the host genome using viral vectors, transcribed by RNA polymerase III.


TRC1 pLKO.1 shRNA transfer vector

Non-target shRNA control plasmid. Modified from: http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/library-information/vector-map.html

• Wide variety of shRNA transfer vectors available

• Vector usually includes:• RNA Pol III promoter (usually U6 or H1

shRNA insert)• regulatory factors• promoters for transcription in packaging

cells• antibiotics for bacteria carrying the

plasmid• Additional components:

• Selection marker (e.g. antibiotic resistance)

• Fluorescent reporter (e.g. GFP)• Transgene

shRNA

• TRC1 library shRNA consructs are available from FuGU:

bacterial glycerol stocks

transfection grade plasmid DNA

Lentiviral particles • for recombinant virus transduction (=infection)


The RNAi Consortium (TRC) shRNA library

• Viral mediated gene (DNA) transfer• Lentiviral Particles

– VSV-G pseudotyped (broad range of mammalian ja non-mammalian host cells)– MINI scale: 1.5 ml virus particles per construct– MIDI scale: 6 ml virus particles per construct– Concentrated Viral Particles: 180 ul / 380 ul, ~ 100-fold concentrated

+ Quality controls: Capsid titer test (p24), internal control

• TRC1 shRNA library constructs • Customers own lentiviral (e.g. shRNA, shRNAmir or cDNA) vectors, e.g. from GBU

• Retroviral Particles– Ecotropic (mouse and rat) and Amphotropic (broader host range) retroviruses available– Moloney mouse leukemia virus (MMLV) and mouse stem cell virus (MSCV) based– MINI scale: 1.5 ml virus particles per construct– MIDI scale: 6 ml virus particles per construct

• Customers own retroviral vectors


Recombinant Viral Particles

FuGU 8January 21st-22nd, 2014

Viral Particle Production & Transduction

1.1 Lentiviral particles are created by co-transfecting packaging vectors + gene transfer vector into packaging cells

1.2 Retroviral particles are produced by packaging cells stably transcribing retroviral packaging

plasmids

2. Viral particles are harvested from the cell culture supernatant

3. Target cells are transduced

• Reasons to concentrate viral particles:– virus p24 titer is usually 104-105 pg/ml (~106 infectious units / ml with HeLa

cells)– particular cell lines require a high viral particle concentration to be infected

(e.g. primary cells)– infections need to be done in a small volume– in vivo infection volumes are extremely small, whereas the viral particle

concentration has to be very high

• At FuGU, lentiviral particles are concentrated utilizing ultracentrifugation– typically a 100-fold concentration is achieved

Concentrated Lentiviral Particles

9January 21st-22nd, 2014 FuGU

• Reasons to purify lentiviral particles– Although sterile filtered, the viral particles may

contain remnants of cells and media

possible inflammatory reaction when the viral particles are used in in vivo applications

Sucrose Purification of Lentiviral Particles


• Virus titer is analyzed to evaluate the quality of the recombinant virus product

• Two complementary methods:– p24 capsid titer test– Live virus titer analysis

• Most of FuGU’s lentiviral products are analyzed with the p24 test

Lentivirus titer testing


• p24 Capsid Titer Test– p24 (MW 24 kDa) is a major structural component of the viral capsid in

recombinant lentiviruses– p24 concentration (pg/ml) is measured from the cell culture supernatant by a

p24-specific ELISA assay– the test measures both functional viral particles and free p24 in the

supernantant the test alone does not give an exact amount of infectious particles

• Live Virus Titer Analysis– constructs containing a fluorescent protein coding sequence (e.g. GFP)– cells are transduced, the percentage of fluorescent-positive (i.e. infected)

cells is calculated and compared to the amount of cells and the volume of viral supernatant used analyzes the number of infectious units in the sample (IU/ml)

Lentiviral Particles - Virus titer testing


• RCV test should always be done before transduced, live cells are moved from BSL II to BSL I facilities!

• Recombinant viruses are replication incompetent, but recombination enabling virus replication is theoretically possible

• Not all viral particles used in infection are transported into cells; the rest stay infectious until removed from the media (e.g. splitting of the cells)

– Biohazard to people – Contamination risk for non-infected cell lines

• Replication competent lentivirus and viral particles can be detected from the infected cells’ media by measuring the concentration of p24 capsid protein

– Samples with a p24 below a certain threshold are regarded as RCV-negative

RCV (Replication Competent Virus) testing


• Managed Biosafety level II laboratory for recombinant virus work for registered customer

– Biomedicum I, 5th floor

• Provides biosafety training and registration

• Viral titer (p24 test) and RCV testing

• Possibility to purchase viral gene transfer products:– packaging cells for lenti- and retroviral particle production– other common cell lines relevant to virus work– transfection reagents

• Materials and reagents found at the BSL II facilities are included in the hourly fee

Biomedicum Virus Core (BVC)


• In qRT-PCR the cDNA amplification can be detected and quantified in real time at each PCR cycle, giving a quantitative measurement of the cDNA accumulation during the PCR

• Quantitative Real-Time PCR (qRT-PCR) services are offered for:– knockdown validation in human or mouse (e.g. TRC1 shRNAs)– validation of ectopic expression (Hs or Mm)– gene expression studies of up to 10 transcripts

• LightCycler® 480 Instrument II and Universal ProbeLibrary (UPL) probes (Roche)

• Service includes:– RNA extraction (on request)– RNA quality control analysis (Bioanalyzer)– primer-probe design– cDNA synthesis– qPCR and data analysis


qRT-PCR Services

• UPL probes are hydrolysis probes similar to TaqMan®– only 8-9 nt long & optimized sequences anneal to multiple sites of the transcriptome specificity is achieved by combining a probe with target-specific primers– contain locked nucleic acids (LNA technology), increased specificity– labeled at the 5' end with fluorescein (FAM), at the 3' end with a dark quencher dye

• 90 UPL probes to cover the whole human, mouse and rat transcriptome

– additional 75 probes for multiple organisms

• Enables multiplex assays: GOI and reference gene– 10 reference gene assays for human, 2 for mouse

• Probes and primers for any assay can be found from Roche’s Universal ProbeLibrary Assay Design Center


qRT-PCR - Universal ProbeLibrary (Roche)


Summary - Products and Services Available

TRC1 shRNA library constructs

shRNA plasmid DNA purification

TransfectionsLentiviral particles

Virus titer testing

Target cell transduction

Selection of infected cells

RNA extraction

qRT-PCR knockdown validation

Data analysis

Results: knockdown efficiency

FuGU’s Service

Customer provided

e.g. knockdown validation service

Step 1 Step 2 Step 3 Step 4

Step 5 Step 6

Step 7 Step 8 Step 9 Step 10

• Public TRC Portal: http://www.broadinstitute.org/rnai/public/• Sigma, MISSION RNAi: http://www.sigmaaldrich.com/life-science/functional-

genomics-and-rnai/shrna/library-information/vector-map.html• FuGU: http://www.helsinki.fi/fugu/index.html• Addgene: http://www.addgene.org/lentiviral/packaging/• jetPEI™ transfection protocol: http://www.polyplus-transfection.com• Universal ProbeLibrary System Technology, Roche: http://www.roche-

applied-science.com


References


Biomedicum Functional Genomics Unit

Contact Information:Email: [email protected]:

Recombinant Virus Services: +358 9 191 25494Genome Profiling Services: +358 50 442 0228

Web pages: www.helsinki.fi/fugu

PersonnelOuti Monni, PhDJuha Klefström, PhDTaru Perttula, B.EngHanna Pesonen, Lab TechnicianMaria Lehtivaara, M.Sc. (Tech.)Pauliina Munne, PhDHanna Ala-Hongisto, M.Sc.Tiina Neejärvi, Laboratory AnalystTopi Tervonen, PhD Minna Lähteenmäki, M.Sc. (Tech.)

Genome Scale Reagents and Services for Functional Studies January 21st - 22nd, 2014 Biomedicum...

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